Protein expression in bacteria and purification

1. The plasmid for recombinant expression of GST-tagged target protein is transformed into E. coli strain BL21 (DE3). A single colony is chosen to inoculate 5 mL liquid LB medium; the culture is incubated at 37 °C overnight (see Note 2). 2. The overnight culture is diluted into 300 mL LB liquid medium to adjust the concentration to OD600 = 0.1; the 300 mL culture is incubated at 18 °C under constant shaking. 3. After about 6 h, check OD600. When OD600 reached to 0.6– 0.8, 0.8 mM IPTG is added to induce protein expression in bacteria; at this stage, also add glucose to a ffnal concentration of 0.2%. The culture is kept shaking at 18 °C (see Note 14).4. The bacteria cells are collected after 12–16 h by centrifugation at 6,000 × g for 10 min at 4 °C. Resuspend in 20 mL GBB buffer and break up the cells by a pressure machine or ultrasonic pulverizer. The supernatant is collected after centrifugation at 12,000 × g for 30 min at 4 °C, and separated into two new 15 mL tubes. 5. Wash 300 μL Glutathione Sepharose 4B beads three times with GBB, and dilute them into the 20 mL protein supernatant. After binding for 2 h at 4 °C, collect the beads by centrifugation at 6,000 × g for 30 s and discard the supernatant. 6. Wash the beads with GST-fused proteins with GBB three times for 10 min by rotating. After the last wash, discard the supernatant completely. 7. Resuspend an aliquot of the beads in 2×SDS protein loading buffer, and heat at 95 °C for 5 min; separate the sample on an SDS-PAGE and include bovine serum albumin (BSA) as reference. 8. Stain the SDS-PAGE gel by colloidal Coomassie brilliant blue (CBB) and quantify the GST-tagged protein by comparing with BSA as reference (see Note 15). 9. Aliquot the beads with GST-fused proteins in 10% glycerol and store at −80 °C.